Coronaviruses (CoV) historically are known to cause relatively mild upper respiratory tract infections, and account for approximately 30% of the cases of the common cold in humans. However, a recently identified CoV, severe acute respiratory syndrome coronavirus (SARS-CoV) causes severe respiratory distress in humans leading to mortality in 9.6% of individuals infected (1). In the year 2003, SARS-CoV established efficient human to human transmission resulting in several super-spreading events. By the end of the outbreak in July of 2003, SARS-CoV was responsible for more than 774 deaths and 8096 cases worldwide involving 29 countries (see World Health Organization website, Epidemic and Pandemic Alert and Response, Diseases, SARs). Since the conclusion of the SARS outbreak several reports of confirmed cases of SARS of unknown origin (29, See World Health Organization website) indicate that the environmental threat of SARS-CoV still exists. SARS-CoV-like virus can be isolated from horseshoe bats in China, and researchers postulate that this is the natural reservoir for the virus (18). SARS-CoV-like virus remains present in intermediate wild animal hosts, such as the Himalayan palm civet, raising the possibility of re-emergence of SARS-CoV infection in humans. Because of the remaining threat, it is prudent to develop effective modalities of pre- and post-exposure treatments against SARS-CoV infection.
During the SARS outbreak, isolation measures proved effective in bringing the outbreak under control. In addition, corticosteroids and antiviral treatments, such as ribavirin, were used to treat infected patients although the efficacy of these treatments for SARS has not been established (5). Therefore, a targeted and effective treatment for SARS-CoV remains highly desirable. In humans, SARS-CoV peak viral load is reached by about 10 days post-infection, thus offering an opportunity for effective post-exposure treatment (6). One modality of treatment that may limit virus replication and thus the spread of the virus is passive immunization with pre-formed neutralizing human monoclonal antibodies (mAbs). Such a treatment during the prodromal phase of the disease could aid in rapid clearance of virus and limit poor clinical outcome and person to person spread, without the adverse effects associated with use of corticosteroids, animal sera, or human sera.
SARS-CoV mediates infection of target cells via the spike (S) protein expressed on the surface. SARS-CoV S protein (Genbank accession number: AY525636; nucleotide sequence SEQ ID NO: 93; amino acid sequence SEQ ID NO: 94) is a type one transmembrane glycoprotein divided into two functional domains S1 (amino acids 15-680) and S2 (amino acids 681-1255) (13). The S1 domain mediates the interaction of the S protein with its receptor, angiotensin-converting enzyme 2 (ACE2) (17). A region of S1 consisting of 193 amino acids forms the receptor binding domain (RBD) which is responsible for ACE2 binding (30). More recently, a receptor binding motif (RBM) within the RBD, consisting of 70 amino acids, has been shown to come in direct contact with the tip of ACE2 (16). The S2 domain of the S protein contributes to infection of the target cell by mediating fusion of viral and host membranes through a conformational change in which two conserved helical regions (HR1 and HR2) of the S protein are brought together to form a six-helix bundle fusion core (11).
The S protein serves as the main antigen that elicits protective immune responses, including neutralizing antibodies in infected humans and animals (3, 4, 6, 9, 12, 14). Intranasal or intramuscular application of a modified vaccinia virus Ankara (MVA) expressing S protein into mice elicits SARS-CoV neutralizing antibodies (3). Immunization of mice with a DNA vaccine encoding the S sequence, devoid of the cytoplasmic domain and/or the transmembrane domain, results in the development of neutralizing antibodies as well as both CD4+ and CD8+ T cell responses (31). However, it is not the cellular, but the humoral (IgG) component of immunity that inhibits viral replication (31). In fact, transfer of immune serum from immunized mice to naive mice reduces SARS-CoV titers following viral challenge (25). Together, these studies show that primarily Abs are responsible for protection against SARS-CoV replication, and indicate the potential therapeutic value of passive transfer of neutralizing Abs against SARS-CoV. The immunogenic property of the S protein, including its ability to induce neutralizing antibodies and its essential role in viral attachment and fusion, make it an ideal target for developing effective immunotherapy against SARS-CoV infection.
During an outbreak, the SARS-CoV can mutate and exhibit antigenic variation. In fact sequence analysis indicated that the clinical isolates could be divided into early, middle, and late isolates (27). The significance of this is demonstrated in the ability of later isolates to escape neutralization by a monoclonal antibody that effectively neutralized an earlier isolate (32). Therefore, it is important to produce neutralizing mAbs that are effective against a wide range of clinical isolates with antigenic diversity. Because of the potential evolution of antigenic variants an effective passive therapy against SARS-CoV will likely contain a cocktail of neutralizing Abs that target different epitopes and/or steps in the entry process, such as blocking receptor binding and fusion.
Passive therapy with human immunoglobulin can confer immediate protection without the deleterious effects associated with the use of animal or chimeric Abs containing animal derived amino acid sequences. Accordingly, there remains an urgent need for potent, broad spectrum antibody therapeutics for use in treating SARS-CoV infection.